Liquid Chromatography—Instrumentation☆

Pilar Campíns-Falcó, Rosa Herráez-Hernández, and Pascual Serra-Mora, Universitat de València, Valencia, Spain
© 2018 Elsevier Inc. All rights reserved.

This is an update of R.P.W. Scott, Liquid Chromatography—Instrumentation, Editor(s): Paul Worsfold, Alan Townshend, Colin Poole, Encyclopedia of Analytical
Science (Second Edition), Elsevier, 2005, Pages 205–213, ISBN 9780123693976.

Introduction 1
Solvent Delivering Units 1
Sample Introduction Configurations 3
Columns 5
Detectors 6
Acknowledgments 7
References 8

Introduction

The history of LC first started in the early 20th century, in the year 1903. That was when the Russian botanist Mikhail Tsvet
discovered a new way to separate plant pigments. In the 1940s, Martin and Synge19 developed a new approach to chromatography,
which used two liquid phases instead of one. The reduction of the packing-particle diameter in the 1960s and the increasing of the
mobile phase velocity through an increased pressure provided shorter analysis times and improved resolution.10,12,13,21,30 From the
above mentioned contributions the groundwork for the development of high performance liquid chromatography (HPLC) laid and
until now new trends have been introduced along the years. The Web of Science data base gives near 420,000 records searching the
topic “liquid chromatography” (period 1952–May 2018), 72,010 of them correspond to the subject “Instruments Instrumentation”
and among them 156 are highly cited papers in the field. From these papers and the new HPLC systems and related products
introduced in 2017–18, the trends in the instrumentation can be outlined.
In reference to the new instrumentation,5 with ultrahigh-pressure liquid chromatography (UHPLC) firmly established as the
modern high performance liquid chromatography (HPLC) platform for more than a decade, all major manufacturers have already
introduced the first- and second-generation of UHPLC systems.1,4,7 In the past few years, manufacturers continued to introduce line
extensions to existing equipment, such as dual-path,23,31 intermediary-pressure, and bioinert systems. New introductions also
appeared to be trending toward application-specific systems, front ends to mass spectrometry (MS) systems, micro- and nano-HPLC
systems,22,32 biopurification systems, portable instruments,18 preparative LC systems,14 and integrated HPLC systems for quality
control (QC) applications. New MS systems abounded,2,9 ranging from single-quadrupole and triple-quadrupole, to quadrupole-
time-of-flight (Q-TOF) hybrid MS systems. Updates to existing chromatography data systems (CDS) proliferated, as well as software
products catering to HPLC method development, peak deconvolution, pharmaceutical development, and stability testing.5
Most standard liquid chromatographs consist of the same components although they may differ in their sophistication level and,
therefore, in their cost.24,25 The basic components are: a solvent delivering unit, a sample injector, a column and a detector, as well
as connective tubing and fittings. Also, modern equipments are connected to a computer for data acquisition and processing.
Modular designs are generally used, so the systems are flexible and can be easily modified (e.g., detectors can be exchanged).
A schematic representation of a typical liquid chromatography system is depicted in Fig. 1.

Solvent Delivering Units

The solvent delivering unit comprises a reservoir for the mobile phase, a degassing device, and a pump with flow and pressure
controllers and connectors (see Fig. 1).
The solvents reservoirs are inert containers of a material compatible with the mobile phase, typically glass. A single solvent
reservoir is used in the simplest systems, but many chromatographs are equipped with two or four containers, so the solvents can be
changed or combined in different proportions. Solvents must be filtered and degassed previously in order to ensure good
reproducibility in flow rates, and also to reduce baseline fluctuations. In-line vacuum degassing is the option used for degasification
in modern LC equipments.
Solvents can be delivered directly from the reservoirs to the pump or to a mixing chamber located between the reservoirs and the
pump (see Fig. 1). In the later configuration solvents from different reservoirs can be combined; the amount of each solvent that is
transferred to the chamber can be controlled electronically, so that the mobile phase can be adjusted to a predefined composition
or, as discussed below, changed along a run. The pump delivers the mobile phase through the column at the desired flow rate.

☆
Change History: September 2018. Pilar Campins-Falco updated the text and references.

Encyclopedia of Analytical Science, 3rd Edition https://doi.org/10.1016/B978-0-12-409547-2.14218-0 1

2 Liquid Chromatography—Instrumentation

Solvent reservoirs (1 to 4)
Vacuum degasser-optional

Solvent mixing valve-optional

Pump: Syringe, reciprocating

(single-, dual piston)

Piston

Autosampler-optional

Sample Metering
vials pump

Injection
valve
Column

Oven-optional
Detector : UV/Vis, MS
(MS/MS), fluorescence,
refractive index, electrical,
evaporative light scattering,
ICP-MS.

Fig. 1 General scheme of a liquid chromatograph.

Pumps are required to deliver the mobile phase at very constant flow-rates and free of pulses. Different types of pumps have been
designed although almost all modern equipments fall into two categories: syringe pumps and reciprocating-piston pumps.
Syringe pumps deliver the solvent by means of a piston which displaces the liquid that fills a chamber at the desired flow rate.
The chamber is typically a cylinder with relative small solvent capacity (<100 mL). The solvent capacity of the chamber limits the
time that the pump is operative until it needs to be refilled. For this reason, unless small flows are applied (<100 mL/min),
reciprocating-piston pumps are a better option. For general applications, reciprocating-piston pumps are much more utilized.
In the simplest configuration, pumps are equipped with a single piston. In single piston reciprocating pumps a motor-driven
cam moves the piston in and out a mixing chamber. When the piston is withdrawn from the chamber the inlet valve opens and the
liquid enters to it and when the piston moves into the chamber the outlet valve opens, so that the mobile phase flows to the column.
The mobile-phase flow rate can be adjusted by controlling the duration of each cycle. Pistons are inert, generally made up of
sapphire, and piston seals are necessary to prevent leaking when the solvent is delivered to the column. It has to be noted that the
flow of solvent passing to the column is intermittently stopped during the first part of the cycle. As a result, a pulsed flow is obtained
which contribute to the baseline noise. For this reason it is preferable a system with two pistons operating exactly 180 degree out of
phase, so that one chamber is filled while the other is emptying. This option results in uninterrupted delivery of mobile phase at
constant pressure. Many systems incorporate a pulse damper to further reduce the pulsations.
In the simplest pumps only one mobile phase at a time can be delivered (isocratic elution). However, for the analysis of complex
samples, the modification of the mobile phase during the chromatographic run may be necessary to achieve suitable resolution.
This is called gradient elution. Gradient elution conditions involve an increment of elution strength along the run according to a
predefined program; if necessary, periods with constant solvent composition can be included in the program. Linear variations of
the mobile phase composition are generally used, but other programs (concave, convex) can be set in some commercial pumps. In
LC, the mobile phase plays a key role, as the solutes interact with both the mobile and the stationary phases and the mobile phase
solubilizes the solute, while in gas chromatography (GC) the mobile phase acts only for the transportation of solutes.
The mobile-phase flow rate can be also modified in the course of the run. The solvent program can be controlled by a
microprocessor or from the computer that controls the system parameters. Gradient elution can be effected in two different ways,
by mixing the solvents prior to the pump (low-pressure mixing) or by mixing them between the pump and the column (high-
 Liquid Chromatography—Instrumentation 3

pressure mixing). In low-pressure systems up to four solvents can be combined (quaternary pumps). These systems only require a
single high-pressure pump to deliver the mobile phase to the column, and the solvent proportions are determined by an electronic
regulated valve system. High-pressure mixing systems require an independent high-pressure pump for each solvent; thus, most of
these systems have been limited to two pumps (binary pumps).
For general purposes flows of 0.5–2.0 mL/min are necessary for separations carried out with conventional columns (4–4.6 mm i.
d., 3–5 mm particle size). Conventional pumps can deliver variable flow rates being the upper pressure limits 400 bar. Preparative
chromatography typically uses flow rates higher than 5 mL/min.
With the implementation of UHPLC pumps capable of generating 1000–1400 bar have been developed (suitable for stationary
phase with <2 mm particle sizes).33
In capillary and nano-HPLC, pumps capable of delivering flow rates <20 and 1 mL/min, respectively are required. Some
equipments have been offered with a splitter (i.e., a tee junction with a restrictor) that in some cases incorporate recycling systems.27
The state of art of portable chromatography is scarce. Several kind of pumps have been proposed in the literature as Table 1
shows. This table also describes some characteristics and troubles of use. Truly portable complete integrated systems are not
available so far.

Sample Introduction Configurations

Sample injection valves, or switching valves, are used to introduce aliquots of the sample into the eluent stream without causing
changes in pressure or flow. Valves consist of a rotor seal with slots that connect adjacent ports and a fixed body with a number of

Table 1 State of the art of portable liquid chromatography

Kind of pump/reference Observations

Gravity pump for ionic chromatography16
Mini-pump together with a pulse damper for
ionic chromatograph6
Electroosmotic pumps (low-medium
pressure)15
Syringe piston pumps17
Syringe piston pumps28,29,36
The system weights 2.5 kg and it can be continuously operated for more than 8 h. Applied to common inorganic
cations (Naþ, Kþ, Csþ, Ca2þ. . .).
Uses open tubular columns, otherwise the mobile-phase does not flow, so it is unpractical for most proposes.
The pump is used at a flow rate of 0.5 mL/min into a portable ionic chromatography equipment. A guard column
instead a conventional column is used to prevent high backpressure. An eluent reflux device is used, so no
wastes are generated and the system is operative up to 27 days. A conductivity detector was used.
An electroosmotic flow is created by introducing Pt wire electrodes at the bottom of two chambers that contain
water; when a voltage is applied an electroosmotic flow is created, so that the stream presses a diaphragm
which discharges the mobile-phase at a flow rate ranging from 0 to 10 mL/min. Water hydrolysis may cause
bubbles and the flow rates depend on the column backpressure. The resulting LC is not well suited for field
tests because it has stringent electrical power requirements.
The pumping system consisted of four programmable microsyringe pumps of different volume options (5, 20, and
100 mL) (LabSmith, Livermore, CA, USA). Two pairs of pumps are connected, to create gradient elution. Sample
is injected by using a 20 nL nano-LC valve, and a 30 cm  100 mm id monolithic RP-18 column was used. The
column was connected to a fused silica with a window and set on an Agilent interface (on-capillary detection).
A LED light source was uses and two photodiodes connected to amplifiers were used for readout. It has not
been tested for field analysis.
Portable LC at different levels of technical performance:
Isocratic pump: The first equipment described used a 24 V DC nanoflow pumping system (engineered by VALCO)
with a 24 mL capacity, which could generate a 100 MPa pressure. During sample loading (effected manually
with a syringe) and pump filling, the pump was stopped. Changing the valve to the injection position directed
both sample (60 nL) and the mobile phase goes to the column at typical flow rates of 74–0.060 mL/min. The
operation of the flow path is controlled by means of an eight-port valve, from the loading/filling to the
dispensing position. Both ends of the column may be connected to the nanopump flow. For detection, an
on-column UV detector was used. The detector is operative for about 10 h. A 15.5 cm  75 mm i.d.
UV-transparent fused silica capillary was used for fabrication of the analytical column, which contained a
monolith stationary phase.
Gradient elution pump similar to that described above, but using two nanoflow pumps connected to a mixer coil
was proposed. The capacity of each pump is 74 mL, and the typical flow rate is 0.35 mL/min. The elution
composition is controlled by means of an actuator, whereas the flow path was regulated by a 10-port high
pressure valve. The same optical setup to that used in the isocratic pump study was used. Example of
application: separation of pesticides (no information on the concentrations assayed)
Ultrahigh pressure nanoflow capillary liquid chromatography: The system was similar to that of the two-
nanopumps (mainly, instead a “Y connection” uses two pumps (30 mL capacity) connected in an injection
chamber). An Orbitrap-MS detector was used. A C18, 100 mm  150 mm id column is used with acetonitrile/
water gradient elution at 2 mL/min. The system is compact although not field portable. It has been applied to
the analysis of peptides.
4 Liquid Chromatography—Instrumentation

external connections. Connections between the different parts of the system (pump, column, waste) can be established by rotating
the valve from the load to the inject positions. The most used valve for LC is the six-ports with an external stainless steel loop with a
predefined internal volume, typically in the 5–1000 mL range (Fig. 2). In the load position the loop is filled with the sample, which
is introduced into the sample loop via a port on the injection valve by means of a syringe. The line of solvent delivered by the pump
is directly connected with the column (see Fig. 1). When the valve is rotated to the inject position the loop is inserted in the solvent
line, so that the sample is pushed by the mobile phase and sent to the column entrance. Valves can be operated manually or
electronically. The loop can be partially filled with the sample by using a precision syringe, although working with the loop totally
filled provides more reproducible results. When the volume of sample injected is very low (<1 mm), internal loops are necessary.
Sample injection can be effected manually but programmable autosamplers that enable unattended analysis are used in many
chromatographs. These devices improve the precision and increase sample throughput. Samples are kept in standard sized vials
sealed with a septum that are placed in trays. The autosampler is programed to withdraw aliquots of samples from the vials
following the desired order. An injection needle that moves in a vertical plane with either a rotating sample tray or articulated arm
perforates the septum and aspirates a defined volume of the sample by means of a highly precise metering pump and fills the
injection loop. By changing the position of the valve the sample is then introduced in the mobile phase flow stream.
The volume of sample injected determines the amount of analyte that reaches the detector and thus, the sensitivity. However, the
injection of a volume of sample excessive results in band broadening. Thus, the injection volume has to be selected according
dimensions and the mobile phase. For samples containing very low concentrations of the analytes, different options can be applied
to increase the detectability as alternative to off-line preconcentration procedures. Among them, on-line in-tube solid phase
microextraction (IT-SPME)26 and on-line solid phase extraction with an extractive column connected on-line with the separative
column are the most successful options.3
In its simplest configuration IT-SPME can be effected by replacing the inert loop of a sample injector by a capillary column
packed or coated with a phase with some affinity for the target analytes, and thus, no extra instrumentation is required. The on-line
SPE with a precolumn often requires an additional pump and valves (optional). These systems are an example of case of
chromatographic separations with coupled columns (or column switching).
In IT-SPME, the analytes are accumulated in the sorbent of the extractive capillary when the sample is loaded with the valve in the
load position; when the valve is rotated the analytes are desorbed by the mobile-phase and transferred to the analytical column for
separation and detection (Fig. 2). The main advantage of this form of SPME is that, although the extraction is not exhaustive, by

(A) Inner loop or IT-SPME capillary

From
pump Column To detector
Injection
valve

(B) Injection
valve
(valve 1) Valve 2

From
pump 1
From
Column To detector
pump 2
Precolumn
(C)
From
Column 1
pump 1
Column 2 To detector

Loop 1 Loop 2

From
pump 2

Fig. 2 (A) Setup for sample introduction in LC with a conventional inner loop and by IT-SPME: load position in solid lines, inject position in dotted lines. (B) Possible
setup for on-line SPE with a precolumn: sample loading in the precolumn, dotted lines in valve 2; transfer and separation of the analytes, solid lines in valve 2.
(C) Possible setup for LC  LC: solid lines, collection of the eluent emerging from column 1 in loop 2 and transfer to the previous fraction from loop 1 to column 2;
dotted lines, transfer to column 2 of the eluent stored in loop 2 and collection of the eluent emerging from column 1 in loop 1. For more details see text.
 Liquid Chromatography—Instrumentation 5

loading of sample volume exceeding several times the internal volume of the extractive capillary sufficient amount of the analytes
can be accumulated, so that a suitable instrumental response can be generated. Therefore, on-line enrichment is effected with no
extra instrumentation. In many applications analyte detectability is suitable without off-line processing of the samples, especially in
water analysis, although this technique has been also proved to be very useful for the simplification of the analysis in other kind of
matrices after a prior treatment (dissolution, lixiviation, dilution). Alternatively, the sample can be passed through the capillary a
number of times until the desired amount of analytes is accumulated in the extractive phase. For this purpose, the sample is
aspirated and dispensed from a vial until the equilibrium is reached using an autosampler. This option is preferable if the volume of
sample available is low, and it has widely used in the analysis of biological fluids.
Initially segments of columns from gas chromatography (GC) separations (typically, of few dm-length) can be set-up as
extractive columns. However, as the number of available phase for GC is relatively small, modified coatings with nanostructure
sorbents and monoliths are increasing used to widen the applications of this technique. Although less used, interfaces specifically
developed to couple HPLC with SPME with fibers have been also developed.
The incorporation to the chromatograph of a valve with more than six ports or two or more six-port valves allows the
combination of two or more column and/or detectors within the same instrument, resulting in very useful and versatile systems
for the analysis of complex mixtures. In these systems the switching valves are used to divert the eluent (or a fraction) coming from a
column to a second column for further separation. This is the basic principle of column switching. Column switching can be used
under many different configurations to increase the separation capacity by coupling columns that operate under different
mechanisms of interactions with the analytes (Fig. 2). A second pump is generally used, so that the two columns can be operated
independently, the only restriction being that the fraction of eluent transferred from the first to the second column must be
compatible with the elution conditions used for the second column. In the simplest configuration only a fraction of the eluent
emerging from the first column is transferred to the second column (cut transfer). This approach is typically used to isolate the target
compounds from the matrix constituents, and thus it is aimed to the on-line sample enrichment and purification. Also, it is used to
increment the selectivity by selecting a second column appropriate for compounds that cannot be resolved in the first column
(e.g., a chiral column).
The most sophisticated column-switching systems are those that operate under a comprehensive configuration (LC  LC). In
these systems cut fractions corresponding to the whole eluent emerging from the first are transferred to the second column that
operates under a different separation mode. This dramatically increases the separation capacity of the system provided that the
separation mechanisms of the two columns are totally independent, as compounds that tend to coelute in the column can be fully
resolved in the second column. The main limitation of this approach is that the time required for the separation in the second
column determines the frequency for transferring fractions from the first column. Separation in the second column must be much
faster. This can be achieved by using a second column much shorter than the first column; systems are designed to transfer a fraction
while collecting the next one as depicted in Fig. 2. Switching valves can also be used to divert the mobile-phase flow rate from one
column to another during method development or to change the detector.

Columns

Nowadays, there are several kinds of particulate and monolithic columns commercially available in many dimensions and phases as
shown in Table 2. Monolithic columns consist of a continuous sponge like skeleton structure with small mesopores and large pore

Table 2 Overview of commercial columns

Company I.D. (mm) Particle size (mm) Phase

Agilent Poroshell 120 4.6–2.1 1.9–4 C18, C8, -CN, HILIC, phenyl-hexyl, -AQ
Poroshell 120 4.6–2.1 2.7 Cyclofructan, hydroxypropylated-b-cyclodextrin, vancomycin, teicoplanin
Chyral
Zorbax 4.6–0.075 1.8–5 C18, C8, -CN, phenyl-hexyl, diphenyl, -AQ, PHA, HILIC PLUS, -NH2, Si-A
Poroshell 300 2.1–0.5 5 C-18, C-8, C-3
BIO-IEX 4.6–2.1 1.7–10 SO3H, COOH, N(CH3)3, N(C2H5)2
Bio-Monolith 5 Monolith Poly(glycidyl methacrylate-co-ethylene dimethacrylate)
ACE ACE 2.1–0.075 2–10 C18, -CN, C18-amide, C18-PFR, C18-AR, HILIC
Phenomenex Luna 4.6–0.3 2.5–15 C18, C8, C5, CN, NH2, PFP, phenyl-hexyl, HILIC, SCX
Kinetix 4.6–2.1 1.7–5 C18, C-8, phenyl-hexyl, HILIC, F5, biphenyl, PAH
Onyx 0.3–0.1 Monolith Monolithic silica road
Chyral 4.6 5 Cellulose, cyclodextrin, penicillamine
Thermo Accucore 4.6–2.1 2.6 C18, C8, C30, phenyl-hexyl, PFP, HILIC, urea/HILIC
Acclaim 4.6–2.1 2.6 C18, C8, phenyl, HILIC, wax, WCX, surfactant
PepSwift 0.5–0.1 Monolith Polystyrene/divinylbenzene
Waters Xselect 4.6–1 2.5–3.5 C18, CN, PFP, phenyl
Peptide BEH 0.3–0.075 1.7 C18, C4
6 Liquid Chromatography—Instrumentation

size. In their constant efforts to increase analytical throughputs, column manufacturers have offered shorter, narrower columns
packed with finer particles.
Reducing the internal diameter (i.d.) for a given particle size provides increased sensibility as can be seen in Fig. 3. Their progress
in designing, manufacturing, and packing particles has resulted in the standard column currently marketed, that provides higher
efficiency and better sample resolution than those of 10 years ago, in a far shorter time. Reversed phase LC, which employs a
nonpolar stationary phase and a polar mobile phase, is the most used mode and habitually employs a C-18 phase (with 3–5 mm of
particle diameter size). The resolution power of modern LC columns is essentially controlled by wall and/or border layer trans-
column eddy dispersion effects, depending on whether the column is radially equilibrated or not. Under a preasymptotic dispersion
regime, the performance of short and wide HPLC columns is controlled by the border effects.8
New methods have been developed for designing novel functional core–shell particles that can improve the efficiency of the
separation for complex mixtures of polar analytes, isomers or structures with similar structure/properties.8,11,34,35 Hydrophilic
interaction phases have emerged as a valuable complimentary option to reversed phases.20
Controlling the temperature during a chromatographic separation may be important to achieve reproducible results because this
variable affects the interaction of the analytes with both the mobile and stationary phases. For this reasons manufactures offer the
option to install the column in a compartment (oven) that keeps the temperature at desired value.

Detectors

The properties of the ideal detector could be stablished like: adequate sensitivity (typical range, 108 to 1015 g analyte/s); good
stability and reproducibility; a linear range for analytes extended to several orders of magnitude; a short response time independent

80
1
1

Absorbance
NanoLC 2 0.5

70 CapLC
0
210 260 310 360
Wavelength (nm)

60

1
2
Absorbance

50
Absorbance (mAU)

0.5

0
1 210 260 310 360
40 Wavelength (nm)

30

20

10
1

0
5 7 9 11 13
Time (min)
Fig. 3 Chromatograms obtained for a mixture of (1) metribuzin and (2) triflusulfuron-methyl by IT-SPME coupled on-line to capillary-HPLC and nano-HPLC. The
figure also shows the normalized UV spectra registered for the two compounds at their respective retention times. Conditions for capillary-HPLC: IT-SPME capillary
coating, tetraethyl orthosilicate (TEOS)-trimethoxyethylsilane (MTEOS) containing SiO2 nanoparticles (NPs), 24 mL internal volume; volume of sample, 100 mL;
mobile-phase acetonitrile–water in gradient mode at 6 mL/min; column, Zorbax C18 (0.5 mm  150 mm, 5 mm particle size. Conditions for nano-HPLC: IT-SPME
capillary coating, TEOS-MTEOS with CuO/SiO2 NPs, 0.66 mL internal volume; volume of sample, 200 mL; mobile-phase acetonitrile–water in gradient mode at
0.5 mL/min; column, Zorbax C18, 0.075 mm  50 mm, 3.5 mm particle size. Concentration of metribuzin, 25 mg/L; concentration of triflusulfuron-methyl, 50 mg/L
and 5 mg/L for capillary- and nano-HPLC, respectively. For other details, see text.
 Liquid Chromatography—Instrumentation 7

of flow rate; reproducible and ease of use; with predictable response for all analytes and nondestructive to the sample, among
others.
The detector may be incorporated in the total chromatograph or connected to the column as a separate component (see Fig. 1).
For preparative work the detector outlet is often connected to a fraction collector. For semipreparative work, this usually consists of a
carousel of collecting tubes that can be alternately and automatically filled by the eluent from the detector outlet.
Table 3 summarizes the fundamentals and some advantages and disadvantages of the main detectors used in combination with
liquid chromatography. Hyphenated techniques refer to the coupling of a separate independent analytical technology to an LC
system. The most common is mass spectrometry (LC–MS), and technologies such as infrared spectrometry (LC–IR) and nuclear
magnetic resonance (LC–NMR) have also been used. The LC–MS will be considered here because of its greater development. For
coupling LC and MS an interface is needed. Atmospheric pressure ionization (API) interfaces are at the moment the most common
due to their versatility and general ease of use. There are several main type of APIs: electrospray ionization (ESI), which is the most
general technique; atmospheric pressure chemical ionization (APCI); atmospheric pressure photoionization (APPI); atmospheric
pressure matrix-assisted laser desorption ionization (AP-MALDI). Systems with conventional and miniaturized LC with API
interfaces and several analyzer geometries are available: magnetic sector (B), quadrupole (Q), ion trap (IT), time of flight (TOF),
triple quad (QQQ), Q-TOF, TOF-TOF, and Q-IT. LC coupled plasma-mass spectrometry (LC–ICP-MS) has progressed to become an
essential tool for studying elements in samples.
Although all the detectors summarized in Table 3 provide relevant qualitative information from the retention time and the
interpretation of the obtained signal, diode array detection (DAD) and MS detectors are remarkable in this sense. As it is given in
Table 3, DAD provides class specific information from the spectra (see Fig. 4).
Fig. 5 shows the options of combining MS analyzers for resolving complex mixtures in terms of separation and class specific
information, compound specific information, even moiety specific information.

Acknowledgments

The authors are grateful to the Generalitat Valenciana (PROMETEO 2016/109) and Spanish MINECO-AEI and EU-FEDER (project
CTQ2017-90082-P) for the financial support received. P.S.-M. is grateful to MINECO/EU for his predoctoral grant (project
CTQ2014-53916-P).

Table 3 Properties of detectors used in liquid chromatography

Kind of detector Fundamentals Observations

Refractive index To measure a bulk property of the mobile phase leaving the Universal, low sensitivity, not compatible with gradient elution,
column: its ability to refract to bend light. Bulk property temperature-dependent
Detector of choice for carbohydrates or in the separation of
polymer by size-exclusion chromatography
UV–vis absorbance: To measure the absorbance of the species Suitable for absorbent species, sensitivity dependent of their
Fixed or variable DAD provides spectra with time molar absorption coefficient, compatible with gradient elution,
wavelength and diode robust and the most used
array detector (DAD) DAD suitable for class specific information
UV–vis fluorescence To detect any compound absorbing and emitting light Selective, high sensitivity
Common uses: amino acids, aflatoxins, vitamins, polyaromatics
Conductivity To measure the ability of a solution to conduct a current when Universal for ionic species. Small, highly charged compounds
placed in an electrical field larger response that large, less charged compound
Widely used in ion chromatography
Electrochemical To measure the ability of a species to undergo either oxidation or High sensitivity: It depends on the extent of oxidation or reduction
reduction: measuring the change in current under a constant that occurs at given potential of the electrode
electric field or the change in the electric field produced when a Destructive detector
constant current is present
Evaporative light To measure the scattering of a beam of light by particles of Universal, destructive technique, does not work well when the
scattering (ELS) compound remaining after evaporation of the mobile phase. sample volatility is similar to the mobile phase
Bulk property For samples that have low or no UV/vis response and do not
ionize well for mass spectrometry: triglycerides, lipids, natural
products, sugars, and oils
Mass spectrometry To measure the mass-to-charge ratio (m/z) of charged particles An interface is needed for LC coupling, selective and high
sensitivity. Suitable for class specific information and
compound specific information, even moiety specific
information from given systems
Destructive detector, expensive. Problems due to signal
suppression
8 Liquid Chromatography—Instrumentation

80

Absorbance (mUA)
60

40

20

400
350 14
12 13
W

11
9 10
av

300
ele

8
7
n

6
gt

250 5
min)
h(

4 (
Time
nm

3
2
)

200 1
Fig. 4 Plots obtained by IT-SPME coupled on-line to nano-HPLC with DAD detection for a mixture of atrazine-desisopropyl (retention time, tr ¼ 5.0 min),
terbuthylazine-desethyl (tr ¼ 6.5 min), terbuthylazine (tr ¼ 9.0 min) and irgarol-1051 (tr ¼ 11 min) at concentrations of 50, 25, 2.5, and 2.5 mg/L, respectively.
Conditions: IT-SPME, 15 cm capillary coated with TEOS-MTEOS with SiO2 NPs (i.d. 0.075 mm), volume of sample processed, 500 mL; mobile phase
acetonitrile–water in gradient mode at 0.5 mL/min; column, Zorbax C18, 0.075 mm  50 mm, 3.5 mm particle size. For other details, see text.

Fig. 5 Possibilities of the combination of MS analysers: Tramp Ionic- MS and MS/MS.

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